Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103

We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 ？ resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.The quaternary structure of Nce103 resembles the typical plant type β-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc containing enzyme which catalyzes a reversible reaction to form bicarbonate from carbon dioxide and water [1,2]. By facilitating the interconversion between CO2 and bicarbonate in vivo, CA activity was proposed to be required to ensure adequate level of CO2or HCO3- as the substrates for other enzymes [3]. Therefore, CAs have been found to play an essential role in a series of fundamental biological processes such as photosynthesis, respiration, pH homeostasis and ion transport [4]. Based on the origins and structural features, CAs are divided into six classes, termed α, β, γ, δ, ε and ζ, respectively [5]. The α-CAs were found in mammals, algae and prokaryotes to facilitate the exchange of CO2 in the respiratory cycle. The β-CAs were characterized in higher plants, algae and prokaryotes, and those in higher plants have been reported to play a critical role during photosynthesis [6]. The γ-class was first identified in the thermophilic archaeon Meth